Hybridoma producing monoclonal antibodies (mAbs), specific for three clinically significant Pseudomonas aeruginosa (serotypes 03, 06, and Oil), were generated to investigate the prevalence of these serotypes in three Northwestern Ohio hospitals. Fusion products reacting with bacterial cells or membrane extracts were detected by enzyme-linked-immunosorbent assay (ELISA). Three mAbs designated: 72C, 11 H (IgM) and HE (IgG2b), were selected. These mAbs reacted with approximately 40% of the clinical isolates of P. aeruginosa in each hospital. The incidence of serotype Oil varied in these hospitals, ranging from 13.2%-23.8%. Serotype Oil predominated in two of the three hospitals. The prevalence of serotype 06 was similar in all three hospitals (13.6-15%). In one of the hospitals (Hospital 2), the occurrence of serotype 06 was slightly higher (15%) than serotype Oil (13-2%). Serotype 03 occurred less frequently (1.5%) in one of the hospitals than in the other two (10-11%). None of the serotypes showed clear predilection toward any body site. The mAbs did not react with other strains of P. aeruginosa, nor with other gram-negative or gram-positive organisms. The results of Immunofluorescence and Western blot correlated well with ELISA. However, ELISA showed a higher sensitivity, indicating the usefulness of this technique for serotyping P. aeruginosa.